mitochondria have been shown to play a critical role in the regulation of the intrinsic pathway to apoptosis through the release of apoptogenic factors 9i.e. cytochrome c) that activate specific class of cysteine proteases (caspases), leading to cell death. During apoptosis, several alterations to mitochondria physiology occur, amongst which is an early change in mitochondrial pH. Many of the changes in mitochondria physiology that occur during apoptosis are thought to take place because of the opening of a pore, know as the mitochondrial permeability transition (MPT) pore. The MPT pore is hypothesized to consist (minimally) of the outer membrane voltage-dependent anion channel (VDAC), the inner membrane adenine nucleotide translocase (ANT) and matrix cyclophilin D (CypD). Members of the Bcl-2 family of proteins modulate the activity of the MPT pore, potentially via direct formation of pores in the outer mitochondrial membrane and/or through interaction amongst themselves or with components of the MPT pore. However, the functional and structural components that constitute the MPT pore in situ, their regulation, and how they interact with each other and Bcl-2 family members, is not known. Recent evidence from our laboratory and those of Reed and colleagues, indicate that Bcl-2 family members modulate apoptosis-induced alterations in mitochondrial pH, and suggest that mitochondrial pH may one of the mechanisms regulating MPT pore activity during apoptosis. Therefore, our specific aims are: 1) Determine whether the MPT pore components interact in a pH-dependent fashion during apoptosis in situ. We will determine whether these proteins interact in situ before or during apoptosis, and if mitochondrial ph regulates these interactions, using our recently developed "two-fusion" FRET technique. 2. Determine the mechanisms by which the anti-apoptotic Bcl-2 (and conversely the proapoptotic Bax-like) family members exert their biological activities. We will investigate whether the pore-forming activities of Bcl-2 family members and/or their ability to interact with specific components of the MPT pore are important in their mechanism(s) of action. In the proposed studies we will test the following hypotheses: 1)Alterations in mitochondrial pH regulate the opening of the MPT pore and cytochrome c release during apoptosis; 2) Bax interaction with the ANT is required for opening of the MPT pore; 3)Pro-and anti-apoptotic Bcl-2 family members modulate MPT pore activity via interactions with the VDAC; 4) Pro- and anti-apoptotic Bcl-2 family members exert their apoptotic regulatory activity by modulating mitochondrial pH, either directly through their pore forming activity or by indirectly by interacting with other family members or with constituents of the MPT pore; and 5) mitochondria contain pH-dependent caspases activity that is required for induction of the MPT pore. These studies are both significant and innovation, as they address the mechanisms of a fundamental biological process, apoptosis, involved in a number of normal and pathological activities of organisms and employ new and novel methods to obtain information that cannot be achieved with other experimental strategies.